Biochemistry I
Lecture 5
[ A ]
[ HA]
f
 pK a  log A
f HA
pH  pK a  log
Buffers:


January 23, 2013
To make a solution at a desired pH
That acts as a buffer, pH=pKa +/- 1
Polyprotic Buffers:
1. Select weak acid based on any of its pKa values.
2. Use pKa closest to desired pH to calculate fHA and fA-.
3. How to make the buffer, select one of the following three
methods:
i) Use chemical forms of “(HA)” and “(A-)” that
represent the species present at the pKa you choose, e.g.
NaH2PO4/Na2HPO4 if the middle ionization was used.
ii) Starting from completely protonated form (e.g.
H3PO4). Add sufficient whole equivalents (n) to reach
the buffer region you are using, add (n + fA-) equivalents
of strong base, or (n + fA-) [AT] moles of strong base.
iii) Starting from completely ionized form (e.g. Na3PO4).
Add sufficient whole equivalents (n) to reach the buffer
region you are using, add (n + fAH) equivalents of acid,
or (n + fAH) [AT] moles of strong acid.
Diprotic buffer example:
O
O
O
O
pH
The diprotic acid shown on the right has
O
O
O
O
pKa values of 3 and 6.
How would you make 0.5 L of a 0.5 M
HO
O
OH
OH
buffer solution at pH=2.5, using only the
disodium salt (Na2A) of this acid?
Step 1: Pick the ionization whose pKa is
closest to the desired pH, thus
defining the pKa value to use for
your calculations.
Step 2: Use that pKa to calculate
10
the fraction of each species,
9
fHA and fA- that will give the
8
desired pH of 2.5:
7
Step 3: Construct buffer using
6
method (iii) from lecture 4
5
notes. The titration of the
4
sodium salt with a strong acid.
3
i) Determine the number of
2
equivalents of strong acid
1
(HCl) to use to reach the
0
correct pH, starting from the
0
0.5
1
1.5
fully deprotonated weak acid.
Equivalents
For a monoprotic buffer this
would simply be fHA.

1
In this case, because it is a diprotic buffer, and we are R  [ A ]  10( pH  pKa) f
HA 
[
HA
]
1

R
starting from the fully deprotonated form, we have to
add one full equivalent of HCl to reach the buffer
1
R  10( 2.53.0)

region that is being used. Therefore:
1.33
eq. of HCl = 1 + 0.75 = 1.75 eq
 0.33
 0.75
ii) The number of moles of the strong acid to use is:
#eq  mol weak acid  vol = 1.75 [AT]V = 1.75  0.5 mol/L  0.5 L = 0.438
mol
1
2
R
1 R
0.33

1.33
 0.25
fA- 
Biochemistry I
Lecture 5
Lecture 5: Amino Acids
January 23, 2013
Reading: Horton 3.1, 3.2, 3.4. Nelson 3.1-3.2
Key Terms





Mainchain atoms
Sidechain atoms
Chiral center
Acidic Residues

Basic Residues
Charge Calculations
O
5A. Structure and Properties:

An amino acid is a carboxylic acid with an amino group.
Most biological amino acids are -amino acids because the
amino group is attached to the -carbon.
 The "mainchain" or "backbone" atoms (N, C, C=O) are
the same in each of the 20 commonly found amino acids.
 The sidechain atoms are unique to each amino acid and
give rise to the unique properties of that amino acid.
 The sidechain atoms are designated with Greek letters,
base on the nomenclature for carboxylic acids.
 The pKa of the carboxylate is ~2.0 and that of the amino
group is ~9.0. Sidechain ionizable groups are also found
on some amino acids.
 Amino acids are joined together to form linear polymers
by the formation of a peptide bond between the carboxyl
of one amino acids and the amino group of the next. This
reaction releases water and is thus dehydration reaction.
The peptide bond can be broken by the addition of water, a
reaction called hydrolysis (hydro-lysis).
Expectations:
 Full name of each (20) amino acid
 3 Letter name of each amino acid
 Structure of each amino acid
 Properties of the side chains:
i) Ionization of groups (pKa)
ii) H-bonding capability
iii) Functional groups (polar/nonpolar)
 UV absorbance, calculation of protein concentration.
O





O


O


+

NH3
CH3
H
H3N
+
OH
H2C
O
H3N
O
O
5B. Chirality & Optical Activity:
In all amino acids (except glycine) the -carbon is chiral. In some amino acids,
additional chiral centers are present. These are chiral centers because all four groups
attached to the carbon are different. This means that the mirror images of these
compounds cannot be superimposed. The two mirror images are called enantiomers.
COOH
COOH
H
H
NH3
H3C
CH3

Enantiomers have the
following attributes:
 Identical physical properties (except rotation of polarized light).
 Markedly different biological properties.

Most common amino acids have an S configuration. An older, but very much
used, notation is D and L. This notation is based on the chirality of a reference
compound and all amino acids that are found in proteins are L.
2
H
+
NH3
O
Biochemistry I
Lecture 5
January 23, 2013
Importance of chirality in Biology: Usually only one enantiomer is active in
biological systems. As indicated above, only L-amino acids are used to make
proteins. Amino acids of the other enantiomer (D) are generally harmless. This is
not always the case for other compounds with chiral centerss:
Thalidomide. This drug was prescribed as a sedative in the late 50s and early 60s.
It was withdrawn because it causes birth defects by interfering with the
development of the baby (teratogens). This activity is associated with only
one enantiomer. The other enantiomer is safe.
Naproxen (Aleve): One enantiomer relieves pain, the other causes liver damage.
H
H H H
O
H
H
H
N
O
N
H
O
O
H
H
Ionizable Amino Acids/Protonated - deprotonated forms
Protonated (pH<<pKa)
Carboxy-Terminus
pKa=2
H2
C
H2N
H
N
O
Amino-Terminus
pka=9
H
H
N
H2
+ C
H
O
Protonated
Aspartic Acid (Asp)
pKa=4
O
Glutamic Acid (Glu)
pKa=4
O
O
H
O
C
H2
OH
Protonated
Histidine (His)
C
pKa=6
O
C
H
C
N
Deprotonated
C
+ N H
N
N
H
H
O
O
C
C
H2
Deprotonated (pH>>pKa)
O
H2
H
N
C
C
O
H2N
H2
O
O
H2
H
H
C
N
C
OH
N
H2
H
O
Deprotonated
O
C
H
N
O
H
Lysine (Lys)
C
pKa=9
O
N
H
O
H
H
Arginine (Arg)
pKa=12
C
H
+
N
N
H
N
H
H
H
N
H
C
+
H C
H
N
N
H
N
H
H
Other sidechain ionizations that are less important: Tyr-OH pKa=10, Cys-SH, pKa=8.
Which groups don’t ionize at
physiological pH ranges?
S
O
Tyr
H
Cys
H
Zwitterions & Charge Calculation:
Zwitterion: a compound that is ionized, but has no net charge.
The overall charge on a molecule as a function of pH can be calculated by:
i) Identify all ionizable groups on the molecule & their charge when protonated and
deprotonated.
ii) Use the known pKa of each group to determine the fraction protonated
qtotal   ( f HA  q HA  f A  q A )
(fHA) and deprotonated (fA-) at the required pH.
iii) Calculate the overall charge by summing the contribution of each group.
Example: What is the net charge on glycine at pH=8?
O
pKa=2
OH
H
H
N
H
O
+
O
H
O
N
H
O
Fraction Protonated
H
H +
pKa=9 N
H
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
3
1
2
3
4
5
pH
6
7
8
9 10
Biochemistry I
Lecture 5
Amino Acid Structures (pH = 6.0)
H 3N
+
H
H
C
H 3N
C
CH 3
H 3N
CH 3
+
+
H 3N
NH
O
O
N
OH
O
+
O
H 3N
Serine
Ser
O
N
H
Tryptophan
Trp
H 3N
H 3N
O
N
O
Lysine
Lys
S
H 3N
O
Aspartic Acid
Asp
O
+
NH3
O
Lysine (Lys)
Polar
Charged
Non-polar
CH 3
pKa = ~ 10
O
H 3N
+
O
NH 2
O Glutamic Acid
Glu
O
Glutamine
Gln
O
O
Methionine
Met
O
+
O
O
+
NH3
+
NH 2
H
+
+
O
O
NH3
Phenylalanine
Phe
O
O
+
+
O
O
OH
Threonine
O Thr
+
O
Asparagine
Asn
H 3N
CH 3
+
O
H 3N
NH 2
O
O
O
Isoleucine
Ile
+
Tyrosine
Tyr
+
O
H 3N
OH
O
CH 3
O
Leucine
Leu
H 3N
CH 3
+
O
O
+
O
H 3N
CH 3
O
Valine
Val
Histidine
His
H 3N
CH 3
O
O
H 3N
+
CH 3
Alanine
Ala
Glycine
Gly
H 3N
O
O
O
O
+
January 23, 2013
+
H
H 3N
H
+
O
H 3N
+
O
O
H
S
O
N
H
NH 2
Tyrosine (Tyr)
Arginine
Arg
H 2N
O
Cysteine
Cys
+
O
Proline
Pro
Polar
Non-polar
Abs UV light
The structure of the 20 common amino acids are shown.
The sidechains are shown in their most likely ionization state at pH6.0,
except His, whose pKa is 6.0 and would be 50% protonated.
Functional Group
Non-polar, non
aromatic
Structure
-CH3 (Ala)
Amino Acids
Alanine, Valine,
Isoleucine,
Leucine
Phenylalanine
Function.
Protein folding – hydrophobic effect.
Binding non-polar drugs.
Serine,
Threonine,
Tyrosine
Aspartic acid
Glutamic acid
H-bond formation to drugs, other
sidechains.
Amide
Aspargine
Glutamine
Amino
Lysine
H-bond formation, donor (NH) and
acceptor (C=O). Note the NH cannot
accept a H-bond.
Usually protonated, pos. charge.
Cysteine
Forms disulfide bonds
Non-polar, aromatic
Alcohol
-OH
Carboxylate
Thiol (sulfhydral)
-SH
4
Protein folding – hydrophobic effect.
Binding non-polar drugs.
Usually ionized, neg. charge
OH